Carbon Isotopes in Individual Compounds

1. Carbon Isotopes in Individual Compounds 03 February 2010

2. GOOD CHROMATOGRAPH SEPARATION

3. Amino Acids - 13C signatures Fatty Acids - lipid biomarkers What Compounds?

4. Diane’s Diagram: Follow the Carbon

5. Background & Fractionation Information for you comprehending pleasure • Mechanistic understanding of the biochemical factors that underpin stable isotope signals • Links biochemistry to stable isotope composition The Idea:

6. Compound-specific isotopes are useful • Different biochemical components can possess different stable isotope values • Structurally similar biochemical components of ecological materials can derive from a range of sources potentially exhibiting different signatures • Biogenic organic matter can change in chemical composition • Reveals contributors mediating processes that would otherwise be masked by in bulk • Biomarkers together with compound specific isotopes information on biological processes • Biochemical components posses significantly different turnover times • Kinetic fractionation can only be determined at the level of the biochemical component and specific pathway

7. Before we can burn our samples up, preparation for compound-specific stable isotopes via GC/C/IRMS follows: Sample Extraction Total Lipid Extract Residue Chromatography Derivatization Hydrolysis Derivatization Free Lipids Monosaccharides & Amino Acids Vaporize!

8. Why so much preparation? Most compounds of interest must be modified, usually of compounds containing polar functional groups, to enhance their volatility prior to GC/C/IRMS injection. Ex: Amino Acid Functional Group Mechanism Reagent Product -NH2 tBDMS MTBSTFA

9. Applications • Fingerprinting • Nutritional Linkages • Biomarkers

10. The Study: Investigation of differences in amino acid metabolism among plants, fungi and bacteria that generate unique patterns of 13C signatures Tool: New approach for tracing amino acid exchange in symbiotic and trophic relationships

11. Lysine and Leucine exhibited significant differences Phenylalanine least variable among taxa

12. Significant difference of non-normalized essential amino acids, distinct isotope clusters Differences in amino acid 13C values between the three most informative essential amino acids

13. Lichen identified as fungi, what role is each organism playing biochemically? Does mostly well identifying what the insects were eating

14. To Summarize: • 13C fingerprinting of amino acids could provide as a powerful in situ assay of amino acid sources in terrestrial ecosystems in • -identifying the primary contributors of amino acids in animals • -understanding symbiotic associations between animals and microorganisms • Greatest accuracy is from the essential amino acids measured based on their more complex biosynthetic pathways

15. Frolicking for food!

16. Carbon values get heavier from south to north by 6 per mil

18. Phe preserves bulk isotope value Phe, Lys, Arg strong correlation to bulk along latitude

19. (2003) The Study: Diets formulated for Pigs to contain 20% protein and wide range in 13C values • The Idea: • Relationship b/w tissue biochemical compounds and diet 13C values • Relationship b/w 13C values of bone collagen and its constituents

20. What do we want to know? (1) Direct incorporation of essential amino and fatty acids (2) Balance between direct incorporation and de novo synthesis of non-essential amino and fatty acids

21. Result for pig on diet 3 Pork Fat Non-essential fatty acids correlated with whole diet values (0.98<r2<0.99). Better than correlation with dietary fatty acid Essential FA Good correlation b/w cholesterol and whole diet d13C values (r2 = 0.81)

22. Essential Fatty Acid: Linoleic Acid Cannot be synthesized de novo; must be incorporated directly from diet. Strong correlation b/w the diet and bone linoleic acid: direct incorporation

23. 13Cvalues of non-essential amino acids were distributed across 10‰, reflecting differences in their assimilation, transport, and biosynthesis. Glycine (serine?) was 8.4‰ more enriched than whole diet values? Also, strong correlation between the stoichiometric and measured bulk collagen values. Estimated 13C values were 1.4‰ more positive than observed values. Study did not include arginine (7.9%) and lysine (4.5%) of carbon to collagen, which are typically depleted in d13C relative to bulk collagen and other amino acids.

24. Amino Acid-Diet Correlations Strong correlation of alanine and glutamate with the δ13C value of whole diet. Decent correlations between essential amino acids (leucine & phenylalanine) and these amino acids in diet.

25. To summarize: • Bone cholesterol and non-essential fatty acid δ13C values correlated well with the whole diet • Bone linoleic acid δ13C values correlated well with dietary linoleic acid • Mass balance calculations using δ13C values of single amino acids accurately predicted the δ13Coh whole collagen • The δ13C values of non-essential amino acids, alanine and glutamate, from bone collagen correlate well with whole diet • The essential amino acids leucine and phenyalanine showed little isotopic fractionation between diet and bone collagen

26. Fatty Acid Routing (Jim et al. 2003 Lipid) Those damn rats!

27. Fatty Acid Routing (Jim et al. 2003 Lipid)

28. Amino Acid Routing (Jim et al. 2006 British J. of Nutrition) Oh Dear God! Not the rats again.

29. C4P/C3E C3P/C4E Amino Acid Routing (Jim et al. 2006 British J. of Nutrition)

30. Amino Acid Routing (Jim et al. 2006 British J. of Nutrition)

31. The Bottom Line(s): • If you want to measure the isotopic composition of bulk diet, use apatite, cholestrol, alanine or glutamate. • If you want to measure the isotopic composition of the lipid component of diet, measure essential fatty acids (e.g. Linoleic Acid). • If you want to measure the isotopic composition of protein, measure essential amino acids (e.g., phenylalanine or leucine), or amino acids that behave as if they are essential (proline). • Routing between dietary protein and bone protein is substantial for animals on protein-rich (20%) diets. It has not been tested for animals on lower protein diets. • Lipid routing is also dependent on the concentration of the particular lipid in the diet.